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Dispersal Flight, Post Flight Behavior and Early Colony Development of the West Indian Drywood Termite Cryptotermes brev...

Permanent Link: http://ufdc.ufl.edu/UFE0022687/00001

Material Information

Title: Dispersal Flight, Post Flight Behavior and Early Colony Development of the West Indian Drywood Termite Cryptotermes brevis (Walker) (Isoptera Kalotermitidae)
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Ferreira, Maria
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: behavior, colony, cryptotermes, dispersal, flight, phototaxis
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The termite species Cryptotermes brevis is a serious structural pest. It is a drywood species spending most of its life inside wood and is only found outside of it when the dispersal flights occur. This important life stage is covered in this study. The major weather cues that trigger the dispersal flights were covered in this study. These appear to be rainfall, humidity, and air pressure. The time of the flights occurred mainly between 1:00 and 2:00 am. In this study, the attraction of alates to light was analyzed. Alates colonized more in areas with higher light intensity than in darker areas. Also, it was shown that the dealates display negative phototaxis, preferring to colonize holes in darker areas, after being first attracted to light. The post flight behavior of the alate including wing release was analyzed. These behaviors were timed, counted for occurrence, and described. The preference for a diameter of colonization site was evaluated, showing that there was a preference for small diameters between 2.3 and 3.3 mm. Early colony development was observed. The time to oviposition was measured, as well as the number of days until each egg hatched. The maximum number of eggs laid per colonizing pair was counted. Observations from this study suggested a method for colonization prevention based on post flight behavior, which was tested. The results were unfavorable and putative reasons for the lack of success are discussed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maria Ferreira.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Scheffrahn, Rudolf H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022687:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022687/00001

Material Information

Title: Dispersal Flight, Post Flight Behavior and Early Colony Development of the West Indian Drywood Termite Cryptotermes brevis (Walker) (Isoptera Kalotermitidae)
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Ferreira, Maria
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: behavior, colony, cryptotermes, dispersal, flight, phototaxis
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The termite species Cryptotermes brevis is a serious structural pest. It is a drywood species spending most of its life inside wood and is only found outside of it when the dispersal flights occur. This important life stage is covered in this study. The major weather cues that trigger the dispersal flights were covered in this study. These appear to be rainfall, humidity, and air pressure. The time of the flights occurred mainly between 1:00 and 2:00 am. In this study, the attraction of alates to light was analyzed. Alates colonized more in areas with higher light intensity than in darker areas. Also, it was shown that the dealates display negative phototaxis, preferring to colonize holes in darker areas, after being first attracted to light. The post flight behavior of the alate including wing release was analyzed. These behaviors were timed, counted for occurrence, and described. The preference for a diameter of colonization site was evaluated, showing that there was a preference for small diameters between 2.3 and 3.3 mm. Early colony development was observed. The time to oviposition was measured, as well as the number of days until each egg hatched. The maximum number of eggs laid per colonizing pair was counted. Observations from this study suggested a method for colonization prevention based on post flight behavior, which was tested. The results were unfavorable and putative reasons for the lack of success are discussed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Maria Ferreira.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Scheffrahn, Rudolf H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022687:00001


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DISPERSAL FLIGHT, POST FLIG HT BEHAVIOR AND EARLY COLONY DEVELOPMENT OF THE WEST INDIAN DRYWOOD TERMITE Cryptotermes brevis (WALKER) (ISOPTERA: KALOTERMITIDAE) By MARIA TERESA MONTEIRO DA ROCHA BRAVO FERREIRA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Maria Teresa Monteiro da Rocha Bravo Ferreira 2

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To my Dad, who was the first to support my love for entomology. 3

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ACKNOWLEDGMENTS I acknowledge and thank Dr. Rudolf Scheffrahn for guiding me on this scientific road, offering an environment that provided cult ivation of knowledge. I also acknowledge the Portuguese Foundation for Science fo r providing me with the funds that allowed me to pursue this degree. I would like to tha nk all the teachers and scientists at the FLREC for helping me increase my knowledge and aiding with any questions that I had. I woul d like to thank the committee members for all the support they ga ve me. I acknowledge Dr. Paulo Borges for initiating my path into the world of termites. I would like to acknowledge my roommates, who provided a relaxing and fun environment at home, but also an intellectually stimulating environment. I woul d like to thank Tini for having the patience to share a room with me for over a year and survive the experience. Finally, I would like to thank my mother and my brother for being a huge support in my life even from far away, and to all my friends back home who supported me anyway that they could from a distance. To all the people that one way or another have contri buted to this work, I gi ve a most sincere thank you. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 CHAPTER 1 INTRODUCTION................................................................................................................. .11 2 DISPERSAL FLIGHTS TIME OF OCCURRENCE AND RELATION WITH WEATHER CONDITIONS...................................................................................................14 Introduction................................................................................................................... ..........14 Materials and Methods...........................................................................................................15 Data Collection................................................................................................................15 Data Analysis...................................................................................................................15 Results.....................................................................................................................................16 Discussion...............................................................................................................................19 3 ALATES POSITIVE PHOTOTAXIS AND DEALATES NEGATIVE PHOTOTAXIS.....22 Introduction................................................................................................................... ..........22 Materials and Methods...........................................................................................................23 Light versus Dark Experiment.........................................................................................23 Experimental process...............................................................................................23 Data analysis............................................................................................................24 Different Light Intensities Experiment............................................................................24 Experimental process...............................................................................................24 Data analysis............................................................................................................25 Negative Phototaxis Experiment.....................................................................................25 Experimental process...............................................................................................25 Data analysis............................................................................................................26 Results.....................................................................................................................................26 Light versus Dark Experiment.........................................................................................26 Different Light Intensities Experiment............................................................................27 Negative Phototaxis Experiment.....................................................................................27 Discussion...............................................................................................................................29 Light versus Dark Experiment.........................................................................................29 Different Light Intensities Experiment............................................................................29 Negative Phototaxis Experiment.....................................................................................30 5

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4 POST FLIGHT BEHAVIOR AND PR EFERENCE OF COLONIZATION SITE DIAMETER............................................................................................................................31 Introduction................................................................................................................... ..........31 Material and Methods.............................................................................................................31 Post Flight Behavior........................................................................................................32 Colony Site Diameter Preference....................................................................................32 Experimental process...............................................................................................32 Data analysis............................................................................................................32 Results.....................................................................................................................................33 Post Flight Behavior........................................................................................................33 Colony Site Diameter Preference....................................................................................34 Discussion...............................................................................................................................35 Post Flight Behavior........................................................................................................35 Colony Site Diameter Preference....................................................................................36 5 EARLY COLONY DEVELOPMENT...................................................................................38 Introduction................................................................................................................... ..........38 Materials and Methods...........................................................................................................38 Results.....................................................................................................................................40 Discussion...............................................................................................................................41 6 EFFICACY OF TWO CHEMICALS IN PARTIALLY TREATED WOOD TO PREVENT COLONY FOUNDATION.................................................................................43 Introduction................................................................................................................... ..........43 Materials and Methods...........................................................................................................44 Results.....................................................................................................................................45 Discussion...............................................................................................................................45 7 CONCLUSIONS.................................................................................................................. ..49 LIST OF REFERENCES...............................................................................................................51 BIOGRAPHICAL SKETCH.........................................................................................................54 6

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LIST OF TABLES Table page 2-1 Average value for occurrence or no o ccurrence of flight for each variable......................17 3-1 Measurements of light intensity (lux) outside and inside the PVC pipe per 10 cm block...................................................................................................................................26 4-1 Mean time in seconds spent on each behavior per termite................................................34 4-2 Mean number of times behavior occurred per termite.......................................................34 5-1 Average time (days) until eggs were laid and larval eclosion...........................................40 7

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LIST OF FIGURES Figure page 2-1 Total number of alates in the water tr ap per day for each year of study between March 27th and June 22nd...................................................................................................17 2-2 Total number of times that flights occu rred for each value of air pressure for the three years of study............................................................................................................17 2-3 Total number of flight occurrences for each value of relative humidity for the three years of the study............................................................................................................. ..18 2-4 Total number of alates in the water trap in 2007 per month and rainfall values in inches for the same period.................................................................................................18 2-5 Total number of alates found in the wate r trap for each hour of the day for the 3 years of the study............................................................................................................. ..19 3-1 Experimental set-up for li ght versus dark experiment.......................................................24 3-2 Total number of holes colonized in lit and dark areas.......................................................26 3-3 Average number of holes colonized per light intensity.....................................................27 3-4 Average number of colonized holes per 10 cm block........................................................28 3-5 Negative phototaxis bioassay.............................................................................................28 3-6 Average number of colonized holes per 10 cm blocks with dark and light.......................28 4-1 Total number of colonized holes for each diameter...........................................................34 5-1 Colonizing chamber showing two larvae, two eggs and a primary reproductive of C. brevis ..................................................................................................................................39 5-2 Image of 3 of the 4 larvae in th e chamber and a primary reproductive of C. brevis .........40 5-3 Coloration of the eggs throughout development................................................................41 6-1 Mean percentage of mortality for treatmen ts with chlorfenapyr, fipronil, and controls for 12.5, 50, and 100% of the surface treated....................................................................46 8

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DISPERSAL FLIGHT, POST FLIG HT BEHAVIOR AND EARLY COLONY DEVELOPMENT OF THE WEST INDIAN DRYWOOD TERMITE Cryptotermes brevis (WALKER) (ISOPTERA: KALOTERMITIDAE) By Maria Teresa Monteiro da Rocha Bravo Ferreira August 2008 Chair: Rudolf Scheffrahn Major: Entomology and Nematology The termite species Cryptotermes brevis is a serious structural pe st. It is a drywood species spending most of its life inside wood and is only found outside of it when the dispersal flights occur. This important life st age is covered in this study. The major weather cues that trigger the disper sal flights were covered in this study. These appear to be rainfall, humidity, and air pressure The time of the flights occurred mainly between 1:00 and 2:00 am. In this study, the attraction of alates to light was analyzed. Alates colonized more in areas with higher light intensity than in darker areas. Also, it was shown that the dealates display negative phototaxis, preferring to colonize holes in darker areas, after being first attracted to light. The post flight behavior of the alate includi ng wing release was analyzed. These behaviors were timed, counted for occurrence, and describe d. The preference for a diameter of colonization site was evaluated, showing that there was a pr eference for small diameters between 2.3 and 3.3 mm. 9

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10 Early colony development was observed. The time to oviposition was measured, as well as the number of days until each egg hatched. The maximum number of eggs laid per colonizing pair was counted. Observations from this study suggested a me thod for colonization prevention based on post flight behavior, which was tested. The results were unfavorable and putative reasons for the lack of success are discussed.

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CHAPTER 1 INTRODUCTION The West Indian termite Cryptotermes brevis (Walker) is a drywood termite belonging to the family Kalotermitidae. It was first described in Jamaica and is a structural pest found globally (except for Asia), infesting buildings and furnitu re, being mainly reported in the tropical and subtropical areas with some is olated occurrences in warmer temperate regions (Light 1934c; Edwards and Mill 1986). It is endemic to Chile a nd Peru where it occurs in nature, away from structural wood (Scheffrahn et al. 2008). As like other drywood termites, C. brevis is a cryptic species which nests in its food source, wood, spending most of its life cycle inside it. C. brevis is a social insect and its colonies comprise three main castes: the reproductives (king, queen, and unmated winged forms called alates); the soldiers; and the immature reproductiv es, soldiers, and false workers or pseudergates (Snyder 1926). Both the male and female reproduc tives remain in the colony as king and queen and may be replaced by secondary reproductives when one or both die allowing the colony to continue. A colony of drywood termites can vary in size from hundred s to a few thousand termites (Nutting 1970) and severa l colonies can be found insi de a single piece of wood. This species has a life cycle which involves a dispersal flight where the alates leave their previous colony in order to form new colonies. The dispersal flights ar e the only occasion when this species is found outside wood (Kofoid 1934). Ot herwise, it never leaves the nest to exploit new food sources (Korb and Katr antzis 2004). After flying, the alates shed their wings and associate as pairs of female and male dealates. These pairs will crawl around in the substrata in tandem, with the male following the female, sear ching for a suitable place to start a new colony (Snyder 1926, Wilkinson 1962, Minnick 1973). The new colony does not produce alates for about 5 years, at which point the colony is considered mature. 11

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The drywood termites are a serious pest acc ounting for about 20% of the budget spent on termite control in the United St ates (Su & Scheffrahn 1990). One of the main methods used for controlling this pest has been th e use of fumigants to eliminat e existing colonies. This method however does not prevent new infestations from o ccurring. Understanding th e dispersal flights of C. brevis is important to develop inn ovative ways of preventing the formation of new colonies. The dispersal flights of termites occur at di fferent times of the year depending on the species. For C. brevis the flight season in South Florida occurs between April and July, with a secondary smaller flight season in November. Some studies have been done on the weather cues that may be involved in the disper sal flights of several termite species, including cues such as air pressure, temperature, relative humidity, rainfall, and wind speed with the occurrence of flights (Minnick 1973, Akhtar and Shahid 1990, Rebello and Martius 1994, Medeiros et al. 1999). Not much work has been done on light attraction for alates of termites although it is commonly accepted that alates are attracted to light when flying (positive phototaxis) and after landing they seek refuge in dark areas (neg ative phototaxis) (Minnick 1973). Also afte r landing, termite alates of some drywood species engage in severa l behaviors including tandem running/crawling, calling and dealation (Nutting 1969). The choice of a colonizing site by so me species of drywood termites that cannot bore into wood involves choo sing a crack or hole of a limited size with enough space to allow turning but small enough to be economically sealed (Nutting 1969). The early colony development is slow for the Kalotermitidae family and numbers are small in the first year for species like C. brevis (Nutting1969). The main objectives for this study were to ex tend the knowledge of the dispersal flight of C. brevis observe and quantify post flight behavior and the early colony development and use this knowledge to improve pest control techniques for this species. With these objectives in 12

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13 mind, this study analyzed: i) what time of the year these dispersal flight s occur, what possible weather cues may be involved in their occurrence and the time of day they occur; ii) when flying, is there a positive phototaxis and does this depend on light intensity, and is this positive phototaxis followed by a period of negative phototaxis after landing ?; iii) once an alate lands, what behavior does it exhibit, how long does it crawl in search fo r a colonizing site, and is there a preferred size for that site?; iv) after a pair is formed and a colonization site is chosen, how long does it take for the first eggs to be laid and for the first la rvae to appear?; v) and finally, with the knowledge of post flight behavior, ca n a lesser area be trea ted with non-repellent insecticides with the same efficien cy as the whole surface treatment?

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CHAPTER 2 DISPERSAL FLIGHTS TIME OF OCCU RRENCE AND RELATI ON WITH WEATHER CONDITIONS Introduction Cryptotermes brevis has dispersal flights at different times of th e year depending on the location in the world where their infestations occu r. These different times of flight occurrences seem to indicate that weather conditions may have a role in cueing the species in to what is the most favorable time to have dispersal flights. The dispersal flight season of C. brevis in South Florida occurs between the months of April and July, with a second sma ller season in November. The occu rrence of dispersal flights of some termites have been correlated with some weather conditions. On a monthly basis the dispersal flights can occur either before or at the end of the rainy seas on (Rebello and Martius 1994), and they can occur at an optimum temperat ure (Akhtar and Shahid 1990). On a daily basis the occurrence of flights appears to be correlated with the air pre ssure, temperature, and humidity (Minnick 1973). The influence of wind speed on th e dispersal flights of termites depends on the species with some showing a strong correlation between low wind speeds and the occurrence of flights (Akhtar and Shahid 1990), while others have no apparent correlation. Observations have been made on the time of day of dispersal flights for C. brevis these being described as crepuscular events (Minnick 1973). Other Cryptotermes species also appear to be crepuscular or nocturnal in their dispersal flights (Wilkinson 1962). The objectives of this study were to analy ze the relationship betw een five atmospheric variables (temperature, rainfall, relative humidity, wind speed, and air pressure) and the occurrence of disp ersal flights of C. brevis and analyze the time of o ccurrence of the dispersal flights during a 24-hour period. 14

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Materials and Methods All experiments were conducted at the Fort Lauderdale Research and Education Center (FLREC), Davie, Florida, in UF building 5031. Th e experiments were conducted in a room of the building partially filled with C. brevis infested wood originated from several infestation sites and kept at ambient temperature and relative humidity, wit hout air conditioning. Data Collection All data were collected between April 2006 and June 2008. The 2006 data was collected by Boudanath Maharajh under the same c onditions as all subsequent data. A water trap was set up consisting of a basi n of 30x19x5 cm filled up to three quarters full with water. A fluorescent light was placed about 20 cm above the basin with an intensity of 1400 lux. The light was left on continuously throughout all the collecting time. All alates found in the basin were counted daily and placed in separate alcohol vials. The data for weather conditions were provided by the Florida Automated Weather Network and by the National Climatic Data Cent er (NCDC). The weather variables used were temperature (C), wind speed (kmph), rainfall (mm), relative humidity (%), and pressure (mmHg) daily averages. In order to determine the time of day the alat es performed their dispersal flight, a Nikon Coolpix S50 point and shoot camera was used. Th e camera was set up facing the water trap and programmed for time-lapse movie mode by taking a picture every 30 minutes. The time-lapse videos were analyzed frame by frame using th e Picture Project soft ware (version 1.6.2 Nikon), and alates in the water trap were counted for every frame. Data Analysis The correlation between the daily flights and the weather cond itions were analyzed with a Principal Components Analysis and a General Li near Model, proc GLM (SAS Institute 2003). 15

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The influence of the weather on the occurrence of flight was an alyzed with a non-parametric Kruskal-Wallis test for all the variables. For this analysis, any data below 1% of the total of alates for that year were considered as a no flig ht so that outlier flights would not influence the results. The time of day the species flies data were analyzed with a proc ANOVA to test if there were intervals of time when the number of alat es flying variance was significantly different and the differences were analyzed by a T ukey grouping test (SAS institute 2003). Results A total of 5,684 alates were collected from the water trap between April 2006 and June 2008. There were two peak flights per flight season per year. In 2006 the peak flights occurred on April 20th and May 2nd, in 2007 the peak flights took place on May 18th and June 14th, and in 2008 the peak flights occurred on May 6th and June 4th(Figure 2-1). The second flight season was not considered because there were only data for 2007. The Principal Components Analysis (PCA) re vealed no correlation between any of the variables and the occurrence of flights. However, when analyzed with a simple GLM, relative humidity showed a significant effect on flight occurrence (p=0.002). The model created by GLM showed a negative effect of relative humid ity although the r-squared value was not high enough to consider the model generated to be the be st fitted model (r-squared=0.04). Using the nonparametric tests however, two patterns emerged showing that there was a significant difference between the occurrence of fli ghts and the occurrence of no f lights that was dependent upon relative humidity and air pressure values. Flights occur significantly more when air pressure and relative humidity were on average 761.2 mmH g and 70.23% respectively (Table 2-1). 16

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Ala t e s in wa t e r tra p (3 ye ar s bet w ee n Ma rc h an d Ju n e ) 0 100 200 300 400 500 600 700 800 9002 7 -Ma r 3 1Ma r 4Ap r 8-Apr 12-Apr 1 6 -Apr 2 0-A pr 2 4-Ap r 2 8 -Apr 2 -M ay 6 -M ay 10-May 14M ay 18M ay 22 May 26 May 30-May 3Jun 7Jun 11-Jun 15-Jun 1 9-Ju ndays of the monthtotal number of alates 2006 2007 2008 Figure 2-1. Total number of alates in the wate r trap per day for each year of study between March 27th and June 22nd. Table 2-1. Average value for occurrence or no occurrence of flight for each variable. For each column variables with the same letter had no significant differences for <0.05. temperature (C) pressure (mmHg) wind speed (kmph) rainfall (mm) relative humidity (%) Flight 24.62a 761.2a 14.42a 4.57a 70.23a No flight 23.79a 762.5b 15.45a 3.81a 73.21b Visually it is possible to see a threshold of air pressure (763.5 mmHg) above which fewer flights occur (Figure 2-2) and the same is visi ble for the relative humidity data (83%) (Figure 23). 0 1 2 3 4 5 6 7 8757 .4 757 .9 758 .4 759 7 59 .5 760 760 5 761 761 .5 762 76 2 .5 763 763 .5 76 4 764 .5 7 65 .1 7 65 .6 766 1 766 .6 767 .1 767 .6 768 .1 76 8 .6 769.1air pressure (mmHg)number of flight occurrences Figure 2-2. Total number of times th at flights occurred for each value of air pressure for the three years of study. 17

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0 1 2 3 4 5 65 0 55 5 7 59 6 1 63 65 67 69 7 1 7 3 75 77 79 81 8 3 85 8 8 90 9 3relative humidity (%)number of flight occurrences Figure 2-3. Total number of flight occurrences for each value of relative humidity for the three years of the study. Observations during 2007 suggest that the fli ghts are dependent on rainfall with higher activity in the months where rainfall is low and lower activity in the months when rainfall is higher with the exception of June (Figure 24). However, no correlation between rainfall and occurrence of flights was found (p=0.47). 0 100 200 300 400 500 600 700 800Ja nu ar y Fe br ua r y M ar ch Apr i l M ay J u ne July Augus t September O c t ob er N o v em b er Dec em b e rmonths of the yearnumber of alates in the water trap0 50 100 150 200 250 300 350 400monthly average rainfall ( mm ) dispersal flights rainfall Figure 2-4. Total number of alates in the water trap in 2007 per month and rainfall values in inches for the same period. There were significantly more flights betw een the hours of 21:00 and 8:00 than during other times of the day, with a significantly high peak betwee n 1:00 and 2:00 am. The lowest occurrence of flights was between 16:00 and 17:00 which was signifi cantly lower than the other 18

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times of the day. Significant differences were also observed for flights occurring between 13:00 and 19:00 hours and the other times of day. (Figure 2-5). gppyy 0 50 100 150 200 250 300 350 400 450 50012: 0 0 13:00 1 3 :0 0 14:00 1 4 :0 0 15:00 15:001 6 :0 0 16:00-17 : 00 17:00-18:00 1 8 :0 0 19:00 19:002 0 :0 0 20:002 1 :0 0 21:00-22 : 00 2 2 :002 3 :0 0 2 3 :00-0 : 00 0: 0 0 1:00 1:00-2:00 2: 0 0 -3 :0 0 3: 0 0 -4 :0 0 4: 0 0 -5 :00 5 : 00-6 :0 0 6 : 0 0 -7 :0 0 7: 0 0 -8 :0 0 8: 0 0 -9 :0 0 9 :0 0 -1 0 :0 0 1 0 :0 0 11:0 0 1 1 :0012:0 0time of da ytotal number of alates b b b b a b b b b b b c c c d dd dd c c c c e time of day Figure 2-5. Total number of alates found in the wate r trap for each hour of the day for the 3 years of the study. Bars with same letter were not significantly different for <0.05. Discussion The water trap data provided proportional in formation on the relativ e number of alates flying the room, even though there was lag between th e actual emergence of the alates from their galleries and the time they land in the water trap, th is lag is short with alat es not flying more than half an hour. The peak dispersal flights of C. brevis occurred between late April and early June in this study. However for each year the flight season shifts a little, with the first peak flight occurring about a month before the second peak. These shifts of the dispersal flight season seem to indicate that there may be weather related cues that trigger the beginning of the flight season. The results of this study show ed that relative humidity and air pressure influenced the occurrence of flights. Both the GLM and the non-parametric tests showed that relative humidity had a negative effect on the occurrence of flights. Williams (1976) argued that C. brevis could thrive in drier conditions in Africa than other sp ecies of the same genus, which may indicate the 19

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effect that lower relative humid ity has for this species. Minnick (1973) also observed that, on an hourly basis, the dispersal flights would occur in relation to the fluc tuations of relative humidity and air pressure, the flights occurring following the lowest air pressure of the day. As for relative humidity the dispersal flights occurred on the low and high peak of relative humidity for that day (Minnick 1973). However, the resu lts of this study indicate th at on a daily basis the flights occurred significantly more when the mean rela tive humidity was 70 % with a threshold of 83% above which fewer flights occurred. Very few flights occurred above the air pressure threshold of 763.5 mmHg. This could be a good indicator as to when dispersal flights may occur as long as the relative humidity is not too high. In the fu ture, studies pertaining to accuracy of dispersal flight predictions based on air pr essure may bring more informa tion about the cues causing the alates of this species to fly. When looking at a whole year, the relationship between the occurrence of dispersal flights and rainfall was conspicuous a lthough not statistically valid. The flights increased at the beginning of the rainy season and at the end with very few flights occurring during most of the season. This is in accordance with other term ites belonging to the Kalotermitidae where this phenomenon has been observed (Rebello and Martius 1994). Although the number of termites flying at the end of the rainy season is not large, it is interesting to see that flights occur when the rainfall drops to values close to 50.8 mm per mo nth. Rainfall seems to be an important cue for the beginning of the flight seas on, with the peak month being the first month with rainfall above 101.6 mm per month, even though on a daily basis the rainfall did not show any influence upon the dispersal flights. However, te rmites were inside wood inside a building so rainfall itself did not influence the dispersal flight season and maybe the changes in the atmospheric conditions due to rainfall acted as cues. 20

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21 Contrary to what Minnick (1973) observed, the time of occurre nce of the dispersal flights happened mainly during the night an d was not a crepuscular event. The water trap captures were most common between 1:00 and 2:00 am. During the months of dispersal f lights, the sunrise and sunset times varied between 5:29 and 6:12 and 18:38 and 19:17 respectively. Thus dispersal flights occurred more at about 2 hours after suns et through about 2 hours af ter sunrise. This is contrary to what Minnick (1973) reported with flights occurring 80 min after sunset and 30 min after sunrise. It is also intere sting to note that alates fly thro ughout the day, but were significantly less common in the middle of the afternoon (betw een 16:00 and 17:00). It would be interesting to investigate other populations to see if time of dispersal flights conc urs with our observations or if population variability could account for the di fferences observed between this study and Minnicks work in Key West, Florida.

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CHAPTER 3 ALATES POSITIVE PHOTOTAXIS AND DEALATES NEGATIVE PHOTOTAXIS Introduction Many arthropods will move toward (positive phototaxis) or away (negative phototaxis) from light. The most common example of nocturnal flying insects that ar e attracted to lights involves moths (Frank 1988). Many nocturnal flying insects are attr acted or confused by lights and this characteristic is often used for catching insects with light traps (Nabli et al. 1999). While most of the castes of termites are nega tively phototacic, the alat es are attracted to lights and seek to emerge into openings and flying into light (Light 1934a). Termites with crepuscular and nocturnal flight s are known to be attracted to lights (Minnick 1973, Sakanoshita and ga 1971, Wilkinson 1962) and light traps are a common tool for capturing alates. Termites flying towards light does not ensu re that colonization wi ll be higher in lit areas, and no studies we are aware of confirm this. Another aspect of the dispersal of C. brevis is that after positiv e phototaxis of alates occurs, the dealates exhibit ne gative phototaxis as observed for this and other species of the genus (Minnick1973, Wilkinson 1962), although no da ta have been produced to confirm this observation. The objectives of this st udy were to analyze if C. brevis alates were attracted to light and colonized more in lit areas than in dark areas, if light intensity had an effect on the number of colonizations, and if there was negative phototaxis in colonization for dealates. The hypothesis tested was that wood with dark areas will be colonized depending upon light intensity as opposed to a control where colonization will occur randomly. 22

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Materials and Methods All experiments were conducted at the Fort Lauderdale Resear ch and Education Center in UF building 5031 in the room described in Chapter 2. Light versus Dark Experiment Experimental process Data for this experiment were collected be tween April and July of 2007. To analyze the difference between colonization in lit and dark areas, 24 transparent plastic boxes (36x23x28 cm) were wrapped in aluminum foil to isolate the light in one box from an adjacent box. The boxes were placed with the cut lid f acing the infested wood and a hole wa s cut on the side (now top) of the box in order to fit the light bulbs (Figure 3-1). The lights were LED Christmas lights (WF model No TS-70) strung in a series of 5 singl e light bulbs attached with tape and hung through the hole. Black tape was used to position the light bulbs in place as well as prevent light from dispersing through the cut hole, so the only light source were in the lit boxes. The lights were randomly distributed with 12 lit boxes and 12 da rk boxes (replicates). The dark boxes had no light bulbs in them. The lit boxes had a light intensity of approximately 40 lux as measured by a light meter (Extech Instruments model No 403125) and the dark boxes had approximately 0.11 lux (due to contaminating light of nearby experiments). A cube of wood (5 cm3) with 6 drilled holes was placed in the center of the box. Each hole was 1.5 cm deep and 2.3 mm diameter and there was a single hole per face of the block. Four thumb push pins were placed on the underside to allow for enough space for the termite to access the hole on the underside. After the dispersal flight season was over, th e blocks of wood were collected and the number of colonized holes was counted per block. A hole was considered to be colonized when a complete fecal seal was present. 23

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Data analysis The data were analyzed using a non-parametric Wilcoxon Matched Pairs test (SAS Institute 2003) to test if the num ber of colonizations in the dark areas and lit areas were different. Figure 3-1. Experimental set-up for light versus dark experiment. One can see the cut lid of the box, the aluminum foil isolating the box a nd the LED Christmas lights above the block of wood. Different Light Intensities Experiment Experimental process These experiments were performed between Ap ril and June of 2008. For the different light intensities experiment, the same 24 boxes used for the previous experiment were used. The LED Christmas lights were also used but this time ther e were four different set ups for the different light intensities with 6 replicat es per light intensity (measured by the light meter): 6 of the boxes 24

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had no light bulbs ( 0.11 lux); 6 boxes had only one LED light bulb with an intensity of approximately 11 lux; 6 boxe s had 5 LEDs together ( 40 lux); and 6 boxes had 10 LEDs attached with an intensity of approximately 480 lux. The boxes were randomly distributed. A block of wood (15x2x9 cm) with 24 holes 1.5 cm deep and 2.3 mm diameter was placed in each box center. After three months, the blocks were collected and the number of colonized holes was counted per block. Colonization was quantified as described above. Data analysis To test if differences between the light intensities were significant, a t-test was used (SAS Institute 2003). Negative Phototaxis Experiment Experimental process In order to analyze the negative phototaxis of the dealates a white PVC pipe, 51 cm long by 7 cm inside diam. was wrapped with black ta pe and closed off on one of the sides. A 102x2x5 cm board was placed inside with 2.3 mm diam. holes drilled 2.5 cm apart on a grid with a total of 40 holes. The outermost holes were 1cm from the edge of the board. Of these 40 holes, 20 were always exposed to light and 20 were exposed to decreasing levels of light towards the closed side of the PVC pipe. The board was visually divided into 10 cm blocks excluding 1 cm at each end of the board which included 4 holes each and m easurements of the light intensity were made outside and inside the PVC pipe (Table 3-1). A board with the sa me dimensions and number of holes was used as control which was completely exposed to light. This was replicated 4 times. The 10 cm blocks were lettered as follows: A) th e first 10 cm from the closed end of the PVC pipe, B) the following 10 cm, and so on, with bloc ks A, B, C, D, E inside the PVC pipe and blocks F, G, H, I, and J outside, with block J be ing the farthest away fr om the PVC pipe. For the controls, the blocks were letter ed the same way as above even though they were all exposed to 25

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light. The number of colonized holes was counted for each 10 cm block using the previously described colonization criteria. Table 3-1. Measurements of light intensity (lux) outside and in side the PVC pipe per 10 cm block outside E D C B A Light intensity (lux) 600 0.70 0.20 0.08 0.04 0.01 Data analysis A Chi-squared test for independence (SAS Institute 2003) was used to test if the distribution of colonizations was dependent on the light intensity. To analyze if the differences between the number of colonizations in each 10 cm block were significant, a t-test for dependent variables was used (SAS Institute 2003). Results Light versus Dark Experiment In the light versus dark experiment a to tal of 43 holes were colonized. There were significantly more holes colonized in the lit areas than in the da rk areas with a p-value <0.0001 (Figure 3-2). 0 5 10 15 20 25 30 35 40 Light Darktotal number of colonized holes ba Figure 3-2. Total number of holes colonized in lit and dark area s. Different letters represent significant differences for < 0.05. 26

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Different Light Intensities Experiment For the different light intensities, a total of 76 holes were colonize d. An increasing number of colonizations were observed with increasing light intensity. There were significant differences between the dark areas and all other light intensities. For the highest light intensity (480 lux) this was not significantly different from 40 lux but was significantly different from 11 lux intensity. The number of colonizations for 11 lux light intens ity was also not significantly different from that of 40 lux intensity (Figure 3-3). 0 2 4 6 8 10 0.11 11 40 480 different li g ht intensities ( lux ) average number of colonizations a b b,c c Figure 3-3. Average number of holes colonized per light intensity. Diffe rent letters represent significant differences for < 0.05. Negative Phototaxis Experiment In this experiment a total of 175 holes were colonized. The controls had no significant differences between the 10 cm blocks (Figure 3-4) and showed that the distribution of colonization was independent of the block (Chi -squared p value: 0.75). The boards placed in the PVC pipes (Figure 3-5) showed that colonization distribution was not independent of the 10 cm block where it occurred (Chi-squa red p value: 0.001). There were decreasing averages of colonizations from the darkest area of the PV C pipe to the lightest area. There were no significant differences between the first blocks A, B, C, and D in the dark area. Block E was significantly different from the firs t four, and not from blocks G, H, I, and J. Block F, the closest 27

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to the PVC pipe, was not significantly different fr om blocks A, B, C, and D and was significantly different from the remainder of blocks (Figure 3-6). 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 ABCDEFGHIJ10cm blocksAverage number of colonized holes Figure 3-4. Average number of colonized holes per 10 cm block. No significant differences were observed for < 0.05 Figure 3-5. Negative phototaxis bioassay. Board inside PVC pipe (top) and control (bottom). Figure 3-6. Average number of co lonized holes per 10 cm blocks w ith dark and light. Blocks A, B, C, D, and E are inside the PVC pipe and blocks F, G, H, I, and J are outside the PVC pipe. Bars with the same letter were not significantly different for < 0.05. 0 0.5 1 1.5 2 2.5 3 3.5 4 ABCDEFGHIJ10cm blocksAverage number o f colonized holes aa a a b a b b b boutside pipe inside pipe 28

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Discussion Light versus Dark Experiment The results for the light versus dark experi ment confirm the hypothesis that colonization occurs significantly more in lit areas than in da rk areas. This shows that wood located in areas that are lit during the night may be more susceptible to infestation by C. brevis. In a world where artificial lights are becoming more and more co mmon these may cause a change of behavior for some species of animals (Longcore and Rich 2004), but the role of artificial lights is beneficial to structure infesting termites like C. brevis In a practical view this attraction to artificial lights puts structures that have wood and that have a continuous light on durin g the dispersal flight season more at risk to being infested than stru ctures that are not lit. On the other hand, this attraction to light can also be us ed (and has been used) to create traps inside structures that are already infested, in order to minimi ze the spread of the infestation. Different Light Intensities Experiment The different light intensities experiment has also shown that there are significantly more colonizations in lit areas than in dark areas as seen before, but it also showed that increasing light intensity does increase the numbe r of colonizations occurring in that area. Minnick (1973) reported differences in the wavelength of light preferred by C. brevis but did not produce any light intensity results as far as light attraction goes. The fact that the middle intensity was not significantly different from the other two shows that the increa se in light intensity causes a gradual increase in colonization by the termites. Again when looked upon from a practical point of view, if an area is lit during the night the mo re intense the light the more susceptible to infestation it will be. On the other hand, if a light trap is to be used to capture C. brevis alates the more intense the light then the more alates it will attract. 29

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30 Negative Phototaxis Experiment Previous experiments with Cryptotermes havilandi (Sjostedt) (Wilkinson 1962) and C. brevis (Minnick 1973) showed negative phototaxis behavior for thes e species in dealates. After landing, the dealates search and colonize darker ar eas. Due to the nature of wood structures, it could be argued that this is not really negative photot axis but that cracks a nd holes that provide a good colonizing focus are usually hidden and in dark areas. However, the present study has proven that negative phototaxis di d occur. That the controls had an independent distribution of colonizations and the semi-shaded blocks did no t have an independent colonization confirmed the negative phototaxis hypothesi s. The lighter block inside the PVC pipe, block E, had significantly less colonizations than the darker blocks A, B, C a nd D which indicated that more colonizations occurred in darker areas th an in lighter areas inside the PVC pipe. However, block F which was 10 cm closer to the PVC pipe showed no significant differences in number of colonizations to the da rker areas inside the PVC pipe. This might have occurred because the termites colonizing that area had searched for coloni zing sites in the dark area all the way and the best sites were already taken by prev ious colonizers inside the PVC pipe. Also they may have landed near the PV C pipe cueing in on the darker area nearby and colonizing the sites near that dark area. However, further studies on this are needed to understand why the 10 cm closer to the PVC pipe on the light side were significantly more colonized than the 10 cm inside the dark PVC pipe where it would be expected considering the negative phototaxis behavior. A hypothesis as to why this happens is that the best holes are already taken. One way to approach this might be using a higher density of colonizi ng holes, so that the number of holes is not a limiting factor.

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CHAPTER 4 POST FLIGHT BEHAVIOR AND PREFEREN CE OF COLONIZATION SITE DIAMETER Introduction The post flight behavior of termites varies according with the species in question. For higher family termites the sequence of behavior al acts are strictly followed while in lower families this sequence is more flexible (Nutting 1969). However some behaviors such as search for a colonizing site, tandem behavior, calli ng, and dealation are common, although in some species like C. havilandi no tandem behavior has been observed (Wilkinson 1962). The pairs of reproductives form after they have landed. After the pair forms, tandem behavior of female leading the male is often observed (Minnick 1973). Although some observations of the post flight behavior of this species have been done, li ttle is known about this behavior, its duration, and frequency of occurrence. Termites often choose a suitable site to start a new colony. Wilkinson determined that for C. havilandi the preferred nuptial site c hosen by termites was a hole in wood that varied between 2 and 3mm, the size was big enough to allow for the termites to turn inside the future copulatorium but small enough that the amount of energy spent sealing it would not be too high (Wilkinson 1962). The objectives of this study were to describe all the behaviors that could be observed that occur after the dispersal flight, time the behavior s and record the frequency that they occur, and determine if there is a preferred hole diameter for colony initiation. Material and Methods All experiments were conducted at the Fort Lauderdale Resear ch and Education Center, in UF building number 5031 in the room described in Chapter 2. 31

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Post Flight Behavior This experiment was run between April and May of 2008. Arenas were set up in order to analyze the post flight behavior of the C. brevis These consisted of white pine boards (18.4x18.4x2 cm) placed on an aluminum tray. The edges of the board were sealed into the aluminum tray with silicone (Master Flow, water based air duct sealant) applied with a caulking gun in order to prevent the termites from crawli ng underneath the board and out of sight. Sixteen 2.3 mm diam. holes 1.5 cm deep were drilled to provide colonizing sites. The trays were placed under a light to attract the alates. An Ap itek A-HD 720p video camera was placed above the arena. Videos were taken between 8:30 pm and 8:30 am and later analyzed using Quiktime software. Three replicates were done. All observed behaviors were described and timed. Colony Site Diameter Preference Experimental process These experiments took place during May of 2007 and May of 2008. To determine if there was a preferred diameter of colonization site, 25 blocks of white pine (10x5x5 cm) were placed under a light for alate attraction. On the surface of these blocks a total of 10 holes were drilled per block. These holes consisted of two holes of e qual diameter per five different diameters. The diameters used were: 1.6 mm; 2.3 mm; 3.3 mm; 4 mm; and 4.4 mm and the holes were randomly distributed on the surface of the block. A hole was considered colonized following the criteria used in Chapter 3. Data analysis The number of colonized holes per diameter si ze was counted and a ttest for independent variables (SAS Institute 2003) was used to test if the number of colonizations were significantly different between the five diameter choices. 32

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Results Post Flight Behavior The behavior of a cohort of 30 termites wa s observed and 10 different behaviors were described as follows: Landing: when termite lands on the substrat e following its dispersal flight. Take off: when termite flies away from the substrate not returning to it. Wing release behavior: Termite twists around for a few seconds sometimes turning on its back and shaking until one to all of the wings are released. Some termites release their wings inside a hole leaving them inside the hole not allowing for the observation of wing release behavior. Hole antennation: termite approaches a hole and uses an tennae to palpate the hole, circles around it and eventually leaves. Crawling: when the termite crawls around the substrata. Entering hole: when the whole body of the termite is inside the hole. Inside hole: termite enters a hole and remains inside for a certain amount of time Exiting hole: when the whole body of termite is outsi de the hole, following being inside the hole. Flickering: succession of quick take off and landi ngs on the substrate, ending on landing on the substrate. Tandem crawling: when two primary reproductives cr awl one in front of the other. The average time spent by a termite on each be havior varied between 9 seconds to 436 seconds (Table 4-1). The occurrence of some of the behaviors did not vary much, with each behavior occurring on average between 1 to 3 times per termite (Table 4-2). Of the 30 termites observed, tandem crawl occurred only once and only 6 pairs were formed inside the holes. All the termites would eventually ente r a hole and not exit it again form ing a pair with a previously occupying termite or remaining in the hole by themselves. 33

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Table 4-1 Mean time in seconds spent on each behavior per termite. behaviors crawling tandem crawl inside hole flickering wing release behavior hole antennation average time (s) 436 368 328 9 15 10 N 30 1 18 6 20 21 Table 4-2. Mean number of times behavior occurred per termite. behaviors holes checked holes entered and exited take off flickering wing release behavior number of occurrences 3 2 1 1 1.4 A typically observed post flight behavior from a termite can be described as follows. A termite lands on the substrate and flicker for a few seconds (sometimes it will take off). After that, it crawls for a few minutes stopping to antenna te up to three holes. It then engages in wing release behavior for a few seconds, enters a hole remains there for some minutes, comes out, and does the same in another hole. If a second termite lands they may engage in tandem crawling for a few minutes and antennate some holes and en ter others until they choose a final hole for colonization. Colony Site Diameter Preference The diameters of 2.3 mm and 3.3 mm had signifi cantly higher occurrence of colonizations than the remainder di ameters (Figure 4-1). 0 10 20 30 40 50 60 70 1.6 2.3 3.3 4.0 4.4 diameter of holes (mm)Number of holes colonized a b b a a Figure 4-1. Total number of colonized holes for e ach diameter. Bars with same letter are not significantly different for <0.05. 34

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Discussion Post Flight Behavior The results obtained in this study for the post fl ight behavior are cong ruent with previous observations with the crawling behavior, tandem behavior and dealati on occurring. No calling behavior as described by Minnick (1973) was observed. The cr awling behavior in search of an opening lasted on average a little over 7 minutes (436 seconds). During this time the termite (or termites if in tandem) antennates some holes or ac tually enters and exits the hole, remaining an average of about 5 and a half minutes inside (328 seconds). This entering and exiting of the hole has often been observed although no reports had been made on how much time was spent on this behavior. The arena design did not, however, allo w for an observation of what happened inside the hole while a termite remained in there. Because only about half of the termites observed actually formed colonizing pairs, it is importa nt to try and understand what happens when a termite enters a colonizing hole. Most of the pa irs were formed by a termite entering an already occupied hole. This behavior is not often observed and begs inte resting questions. For instance, does the termite inside the hole leave any chemical cue to signal other te rmites that the hole is occupied? And if so, is it a repellent cue to indi viduals of the same sex? More research on this matter is needed. The wing release behavior has been observed for C. brevis although Minnick (1973) did not give a detailed description. Th e turning of the termite on its ba ck as it shakes helps the final release of the wings. Dealation took on average 15 seconds which is more time than reported for C. brevis (Minnick 1973) or C. havilandi (Wilkinson 1962) where both reports stated that the behavior is too quick to obser ve, and lasted less than one second. The behavior however was reported to occur mostly out of sight due to ne gative phototaxis so it is possible that the conditions of the arena allowed for this behavior to be fully observed during this study. 35

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The fact that tandem behavior was observed only once is somewhat surprising because this termite species is known to display this type of behavior (Minnick 1973). Ho wever, it is possible that the number of colonizing sites available may have helped in decreasing the occurrence of this behavior. With many holes available, perh aps termites tend to spend less time in searching behavior, finding a hole rather quickly and maybe decreasing the probability of encountering another termite on the substrate and initiating the tandem behavior. Further experiments with fewer colonizing sites available may help in understanding the low occurrence of tandem behavior. It may also show if the number of available colonizati on sites influence the amount of time spent crawling in search of a hole. A question that remains unanswered however is how far a dealate will crawl in search of a colonizing site. Due to some soft ware problems the images obtained from this experiment were not usable for analyzing the distances covered by the termites. Howeve r the technology is now available and further research s hould be done on this subject. Colony Site Diameter Preference As for the experiments of colony site diamet er preference, the results obtained were congruent with previous results obtained for this species although no statisti cal analysis had been performed before (Guerreiro et al. 2007). These results were also congrue nt with those found for C. havilandi (Wilkinson 1962). It has been shown for both species that there is a preferred diameter for colonization that is located between 2 to 3 mm. This was conf irmed with the present study, with the preferred diameter s being 2.3 and 3.3 mm which is why for other experiments in the present study 2.3 mm diam. was used. This continues to support the hypothesis that colonizing pairs prefer a size of hole that allows for enough space to turn around but small enough to be economically sealed (Wilkinson 1962). Another reason for avoidance of bigger diameter holes is that the time spent in seal ing these may take too long leaving the termites 36

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37 vulnerable to predation. Also, smaller voids may be easier for maintenance of ideal conditions to start a colony, with the volu me of the cavity having an e ffect on conditions like relative humidity. This information may be important in that th e diameter preferred is rather small and any small crack or hole in a piece of wood is suscepti ble to colonization and the sealing of these may be necessary in order to decrease the probability of having a colony founded, especially in areas with high nocturnal flight intensity.

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CHAPTER 5 EARLY COLONY DEVELOPMENT Introduction Drywood termite colonies are usually small with only hundreds to a few thousands individuals per colony (Nutting 1970). This is in contrast with higher termites which can have millions of individuals in their colonies. The development of a drywood colony is slow and a colony may not be mature for at least 5 years, when all the castes are present including the reproductive alates (Nutting 1969). Observations have been made on the early development of C. brevis s colonies showing that the development of this spec ies is slow with few eggs bei ng laid and with a pause between the laying of a batch of eggs as long as 5 months. Also, after the first year a colony may have as few as 3 to 4 individuals (Nutting 1969). Aside fro m these observations very little has been done relative to understand ing the early development of a colony of C. brevis The objectives of this study were to incr ease the knowledge of ea rly colony development for C. brevis analyzing the number of eggs laid, the time it takes for oviposition, how long is the incubation period, and how many larvae can a batch of eggs produce. Materials and Methods All experiments were conducted at the Fort Lauderdale Resear ch and Education Center in UF building 5031 in the room described in Chap ter 2. The early development of the colony was observed for a minimum of 30 days and a maxi mum of 100 days, between March and June 2008. To study the early colony development nupt ial chambers were constructed. These consisted of a block of white pine 8x4x2 cm w ith a 2.3 mm diam. hole drilled on the upper side through to a 2 cm diam., with th e larger area 1.5 cm deep and the smaller area 0.5 cm deep. The wider hole was made using a wood drill. These bl ocks were placed on a 9 cm diam. Petri dish 38

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with the larger diameter facing down. The blocks were kept in place by applying silicone (Master Flow, water based air duct sealan t) to the edges of the wood boa rd with a caulking gun. Fifteen blocks were left to be colonized with the smaller diameter facing up, and placed under a light. After two termites occupied the chamber, the bl ocks were removed from the light source and placed with the Petri dish side up and covered with a black plastic bag. The chambers were then checked daily for eggs and larvae (Figure 5-1) with the help of a hand-held magnifying glass (10x). The blocks were kept in the afore men tioned room without controlled temperature or humidity in order to simulate the beginning of a colony in an attic (where many infestations occur) without air conditioning. Figure 5-1. Colonizing chamber s howing two larvae, two eggs and a primary reproductive of C. brevis 39

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Results Of the 15 blocks placed under the light, only 12 were colonized, each was occupied by 2 or 3 dealates and of these 12 only 9 produced active egg laying pairs. A maximum of 5 eggs and a maximum of 4 larvae were observed per colonizing pair (Figure 5-2). Figure 5-2. Image of 3 of the 4 larvae in the chamber and a primary reproductive of C. brevis The average time (days) for each egg to be la id and each larva to eclode are depicted in Table 5-1. The average incubation time of an egg was 54.5 days. The eggs went through a change in color as development proceeded st arting out pink and getting increasingly white towards the time of eclosion (Figure 5-3). No cannibalism of the eggs was observed. Table 5-1. Average time (days) until eggs were laid and larval ecl osion. Numbers with indicates single observations. 1st egg 2nd egg 3rd egg 4th egg 5th egg 1st larva 2nd larva 3rd larva 4th larva Incubation Average number of days 13.4 17.7 24.8 34 63* 68.5 71.5 86* 99* 54.5 40

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Figure 5-3. Coloration of the eggs throughout development. A) pink young egg, B) white egg near eclosion. B A Discussion The results obtained in this study are consistent with the previous finding that drywood colonies are slow developing in early stages (Nutting 1969). Of all the initial colonies observed only one pair produced a 5th egg with all other pairs producing a maximum of 4 eggs. Also in the 100 days of observations only one pair produced 4 larvae from the eggs and no further egg lay was observed. This pause between oviposition ha s been previously observed for this species (Nutting 1969) and is also observed for other termite species ( Coptotermes formosanus (Shiraki)) where cycles of oviposition have been observe d (Raina et al. 2003). The fact that only a maximum of 4 eggs was laid is in agreement with observations of only 3 to 4 individuals within the first year of colony development for C. brevis (Nutting 1969). The queens of C. brevis are not physogastric and as such will not lay as ma ny eggs as larger queens in higher termites (Snyder 1926). This may account for the small num ber of eggs laid per batch and the slow development observed for drywood termites (Nutti ng 1969). Also maybe adults can only supply food for 4 larvae at a time until the larvae reach the pseudergate stages. Egg incubation times are slightly faster than previously observed, with the termites in the present study taking and average of 54.5 days of incubation while previous ly the incubation took 41

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42 between 75 to 81 days (Nutting 1969). Possible expl anations for these disc repancies include: first the type of wood used may differ and this may ha ve an influence on the amount of protein that is provided by the mother to the egg and eventually the sl ower or faster development of the egg. Also conditions of temperature a nd humidity have an effect. Ob servations were conducted under laboratory temperatures (Nut ting 1969) while the colonies in the present study were maintained at room temperature (average 25 C) which was not controlled and varied with the normal weather variations, as well as the relative humidity. This may also have had an impact on the period of incubation. Lastly, th e different results may simply be due to the use of different populations and there may be some variations in the incubation periods between populations. For C. havilandi for example, the population studied by W ilkinson (1962) had an incubation period much more similar to the one found here with a mean of 53 days even though it is a different species. No cannibalism of eggs was observed for thes e colonies, although it is often reported that the primary reproductives will often consume th eir own eggs even with adequate nutrition (Nutting 1969).The coloration of the eggs for C. havilandi started out semi-transparent and became white later. For C. brevis the initial color was pink, but just as for C. havilandi the eggs became an opaque white, which is also the color of the newly hatched larvae. Further studies may be necessary to determine what components are present in the egg at the different stages and what causes the color change. Also a more prolonged study of the colony development is needed in order to better understand the population dynamics of a C. brevis colony.

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CHAPTER 6 EFFICACY OF TWO CHEMICALS IN PA RTIALLY TREATED WOOD TO PREVENT COLONY FOUNDATION Introduction It has been observed that alat es are attracted to light and after they land near the substrate they engage in tandem behavior where the female leads the male in search of a colonizing spot (Snyder 1926, Wilkinson 1962, Minnick 1973). This s earch for a colonizing spot can last for some time and the alates can cover a large su rface area (personal obs.) Because the dispersal flight season is the only time drywood termites are naturally outside the wood, this is a suitable time for termiticide exposure, especially in preventing new colony establishment. The chemicals that were tested in this study were of two different classes. The first was chlorfenapyr which is an aryl-substituted cyanop irrole. This chemical was first synthesized in 1988 (Treacy et al. 1994) and it has been used for agricultural pest contro l purposes. It has been registered for house-hold pests since 2001, but little work has been done in termites (Rust and Saran 2006). The second chemical was fipronil, which is an N-phenylpyrazole. This chemical was first marketed in the USA in 1996 (Cox 2005). It is an insecticide used for a variety of insects, from cockroaches (Zhao et al. 2003), fire ants, to crop pests such as coleopteran larvae, lepidopterans, and orthopterans (Hainzl and Casida 1996). It is also widely used for the control of subterranean termites and its efficacy against these has been well demonstrated (Hu 2005, Saran and Rust 2007). Considering the behavior of the dealates in this study the hypothesis underlying this section of the study was that th e dealates searching behavior would increase the likelihood of contact for dealates with a non-repellent insecticide that is app lied on the wood surface. Also, the mortality would be the same whether the entire surface was treated or when only portions of the 43

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surface were treated. Chlorfenapyr was hypothesized to be as efficient as previously well-tested products containing fipronil. With this in mind, the objectives for this st udy were to determine if treating only a part of the surface area with chlorfenapyr woul d be enough to prevent colonization by C. brevis using bioassays which represent infested areas that include inaccessible portions, evaluate the efficacy of chlorfenapyr as a colonization preventative and compare the efficacy of chlorfenapyr and fipronil (an industry standard). Materials and Methods All experiments were conducted at the Fort Lauderdale Resear ch and Education Center in UF building 5031 in the room described in Chap ter 2. The bioassays were assembled between May and June of 2007. A total of 35 white pine wood boards were us ed in this experiment. The surface area of each board was 134 cm2 (18.4 x 18.4 cm) and 2 cm thick. On the surface of these boards sixteen 2.3 mm diam. holes were drilled equally distan ced from each other to a depth of 1.5 cm. The control boards had no treatment at all and consiste d of 5 replicates. As for the treatments, two surface treatments were used with three treatm ents. A chlorfenapyr surface treatment was applied to the whole surface of the board, 50% of the surface and 12.5% of the surface. Each of these treatments had 5 replicates. The same proce ss was used for fipronil, where for 5 replicates each with 100%, 50%, and 12.5% of the surface treat ed. Both the chlorfenapyr (Phantom, BASF) and fipronil (Termidor, BASF) were used at label rate of 0.125% (A. I. 0.0021g/cm2) and 0.06% (0.0017g/ cm2) respectively. To simulate the worst case scenario for a homeowner, the holes on the boards with whole surface treatment were drille d after the treatments were applied. This was intended to simulate a person that applies the tr eatment and then drills new holes which can be new entry points for alates. The boards were then placed in aluminum trays so that they could be 44

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isolated from each other and placed under a light in for alate attraction. When the holes in the boards were colonized, the trays were removed fr om light and disassembled one week later. All the live and dead termites were recorded and a t-test for independent variables (SAS Institute 2003) was used to assess the differences between the percentage mortality in the different treatments. Results A total of 1,356 dealates were documented bot h in the chambers cr eated for them and on the surface of the boards. All the controls had a significantly lower percentage of mortality than both the chlorfenapyr and fipronil treatments with less than 10% of mortality (Figure 6-1). None of the treated boards showed 100% mortality. The differences between the treatments of chlorfenapyr and fipronil when the boards were treated for 12.5 or 50% of the surface were not significantly different from each other. On the other hand, in the whole (100%) surface treatment, there were differences between chlorf enapyr and fipronil. Fipronil had significantly more mortality for the whole surface treatment wh ile chlorfenapyr was not significantly different for the whole surface treatment comp ared to the other treatments. Discussion Prevention of colony foundation for C. brevis has been studied before with many different chemicals tested (Scheffrahn et al. 1998, 2001). C. brevis has been found to have as much as 2 founded colonies with brood in a total of 22 nupt ial chambers (Scheffrahn et al. 2001). This shows that investment in colony prevention is important because if 9% of nuptial chambers produce brood and possibly develop into coloni es, it is a high enough number to consider prevention. The hypothesis underlying this study is the possible use of le ss insecticide obtaining the same results as with a larger am ount which can be more user-friendly. 45

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Average mortality in treatments0 10 20 30 40 50 60 70 80 90 12.5 50 100surface treated (%) % mor t a lit y Chlorfenapyr Fipronil controls aa b b c a,b dd d Figure 6-1. Mean percentage of mortality for trea tments with chlorfenapyr, fipronil, and controls for 12.5, 50, and 100% of the surface treate d. Bars with the same letter are not significantly different for <0.05. From the results obtained in the present study, it was seen that chlorfenapyr mortality was below 50%, never achieving a high le vel of efficacy. Several explana tions can be considered for these results. First, it is possible that chlorfena pyr is not effective against termites. However, its efficacy has been demonstrated against subt erranean termites where mortality for 500 ppm chlorfenapyr showed 100% mortality after 5 days of e xposure (Shelton et al. 2006). Concentrations as low as 10 ppm (Rust and Saran 2006) resulted in >70% mortality for subterranean termites. These stud ies were performed with workers of subterranean termites and the effect of chlorfenapyr on primary repr oductives is unknown. The dealates have a welldeveloped pigmented chitinous exoskeleton wh ile workers and nymphs have a thinner 46

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unpigmented exoskeleton (Light 1934b). The thic ker exoskeleton may provide some protection from absorbance of chlorfenapyr. Ot her arthropods have been shown to be less susceptible to this particular chemical (Herron and Rophail 2003) although for unknown reasons. Also the dealates may not transfer the toxicant from an exposed nestmate to an unexposed nestmate as it has been seen for Incisitermes snyderi (Ferster et al. 2001). Another consideration is that chlorfenapyr is considered a slow acting insecticide (Moore and Miller 2006) being an inhibitor of oxidativ e phosphorylation, preventing the formation of the crucial energy molecule, adenosine triphosphate (ATP). The effects of this may take longer to act and high mortality may not have been observed because enough time had not elapsed between the contact with the chemical and eventual d eath. Some mortality was always observed and it was significantly different from the controls, thus showing that chlorfenapyr has some effect. Fipronil on the other hand, caused high mortalit y when the whole surface was treated. This indicates that the efficacy of fipr onil is higher than chlorfenapyr. Firponil has acute toxicity after only 24 h for termite workers (Ibrahim et al. 2003) and this may be why it shows better results for the whole surface treatment than chlorfena pyr. Although this mortality was only slightly above 70%, it does not support the hypothesis that the crawling behavior would be enough for the termites to come in contact with the non-repellent chemical. The fact that mortality was significantly higher for the whole surface treatmen t suggests that when there are areas of the surface left untreated, the proportion of survivin g termites, increases significantly leading to more colonizations than with w hole surface treatments. Perhaps the crawling behavior is too fast and the contact with the chemical is too short to provide enough contact for the chemical to cause 100% mortality. Also some termites may not crawl as much as others or may not even come into contact with the chemical at all, wh en only part of the surf ace is treated. New studies 47

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48 with higher concentrations or different formulations that are more easily dislodged, like microcapsulation, may help resolve this question, because it may allow for greater mortality even if the contact is very brief. This may shed so me light on whether the low mortality for partially treated wood is due to the limited contact du ring the crawling behavior in itself or the inefficiency of the chemical.

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CHAPTER 7 CONCLUSIONS From the work done in these studies several conclusions can be drawn about the dispersal flights of the species Cryptotermes brevis The dispersal flights of this species occur in South Florida between April and June, w ith most of the alates flying during the night hours with a peak between 1 and 2 am. Several weathe r cues seem to be i nvolved in triggering these flights, with rainfall being an apparent cue to the start of the flight season on a monthly basis. On a daily basis, lower relative humidity and lower air pressu re seem to be the cues to trigger dispersal flights. When flying, the alates are attr acted to lights and will colonize more in lit areas than in dark areas with increasing col onization occurring with increasing light intensity. After the alates land they demonstrate a negative ph ototacic behavior, colonizing in darker areas as opposed to lighter areas. The alates of C. brevis showed a variety of post flight behaviors which include crawling in search of colonizing sites, a wing release beha vior upon which they release their wings becoming dealates. Also they will antennate several col onizing sites, and enter the site. When a second termite is present they engage in tandem crawling. When searching for a colonizing site they showed a preference for small diamet er openings between 2.3 and 3.3 mm. After choosing the final colonizing focus the pa irs started a new colony with few (4 to 5) eggs during the initial stage. These eggs will in cubate for about 54 days after which the larvae hatched with a maximum of 4 larvae observed pe r pair within the firs t 4 months of colony development. Finally, even though the termites engaged in crawling behavior when landing on a substrate, this was not enough for a termite to have adequate contact with a non-repellent 49

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50 insecticide long enough to cause high mortality. This makes surface treatments that do not reach the entire surface less effective in the preventi on of colonization by this species. There is still much work that can be done on the colonization phase that can suggest new methods of control for this damaging pest.

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LIST OF REFERENCES Akhtar, M. S., and M. M. Shahid. 1990. Impact of rainfall, atmos pheric temperature and wind speed on swarming of termites (Isoptera). Pakistan J. Zool. 22: 65-79. Cox, C. 2005. Insecticide factshee t fipronil. J. Pest. Reform. 25: 10-15. Edwards R., and A. E. Mill. 1986. Termites in buildings, their biology and control. Rentokil Limited, East Grinstead, 261p. Frank, K. D. 1988. Impact of outdoor lighting on moths: an assessment. J. Lepidop. Soc. 42: 6393. Ferster, B., R. H. Scheffrahn, E. M. Thoms, and P. N. Scherer. 2001. Transfer of toxicants from exposed nymphs of the drywood trmite Incisitermes snyderi (Isoptera: Kalotermitidae) to unexposed nestmates. J. Econ. Entomol. 94: 215-222. Guerreiro, O., T. G. Myles, M. Ferreira, A. Borges, and P. A. V. Borges. 2007. Voo e fundao de colnias pelas trmitas dos Aores, com nfase na Cryptotermes brevis. Pp 29-46. In P. Borges, T. Myles [eds.]. Trmitas dos Aores. Principia, Estoril. Hainzl, D., and J. E. Casida. 1996. Fipronil insecticide: Novel photochemical desulfinylation with retention of ne urotoxicity. Proc. Nat. Ac. Sci. 93: 12764-12767. Herron, G. A., and J. Rophail. 2003 First detection of chlorfena pyr (Secure) resistance in two-spotted spider mite (Acari: Tetranychidae) from nectarines in an Australian orchard. Exp. App. Acarol. 31: 131-134. Hu, X. P. 2005. Evaluation of efficacy and nonrepellency of indoxacarb and fipronil-treated soil at various concentrationsand thickness against two subterranean termites (Isoptera: Rhinotermitidae). J. Econ Entomol. 98: 509-517. Ibrahim, S. A., G. Henderson, and H. Fei. 2003. Toxicity, repellen cy, and horizontal transmission of fipronil in the Form osan Subterranean Termite (Isoptera: Rhinotermitidae). J. Econ. Entomol. 96: 461-467. Kofoid, C. A. 1934 Biological backgrounds of the termite problem. Pp1-12. In C.A. Kofoid, S. F. Light, A. C. Horner, M. Randall, W. B. Herms and E. E. Bowe [eds.]. Termites and Termite Control. Univ. Califor nia Press, Berkeley, CA. 795p Korb, J., and S. Katrantzis. 2004. Influence of environmental conditions on the expression of the sexual dispersal phenotype in a lower termite: implications for the evolution of workers in termites. Evol. Develop. 6: 342-352. Light, S. F. 1934a The constitution and development of the termite colony. Pp 22-41. In C.A. Kofoid, S. F. Light, A. C. Horner, M. Randa ll, W. B. Herms and E. E. Bowe [eds.]. Termites and Termite Control. Univ. California Press, Berkeley, CA. 795p 51

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Light, S. F. 1934b. The external anatomy of termites. Pp 50-57. In C.A. Kofoid, S. F. Light, A. C. Horner, M. Randall, W. B. Herms and E. E. Bowe [eds.]. Termites and Termite Control. Univ. California Press, Berkeley, CA. 795p Light, S. F. 1934c Dry-wood termites, their classi fication and distribution. Pp 206-209. In C.A. Kofoid, S. F. Light, A. C. Horner, M. Randa ll, W. B. Herms and E. E. Bowe [eds.]. Termites and Termite Control. Univ. California Press, Berkeley, CA. 795p Longcore, T., and C. Rich. 2004. Ecological light pollution. Fr ont. Ecol. Environ. 2: 191-198. Medeiros, L. G. S., A. G. Bandeira, and C. Martius. 1999. Termite swarming in the northeastern Atlantic rain forest of Brazil. Stud. Ne otrop. Fauna Environm. 34: 76-87. Minnick, D. R. 1973. The flight and courtship behavior of the drywood termite, Cryptotermes brevis J.Environ. Entomol. 2: 587-591. Moore, D. J, and D. M. Miller. 2006 Laboratory evaluations of in secticide product efficacy for control of Cimex lectularius J. Econ. Entomol. 99: 2080-2086. Nabli, H., W. C. Bailey, and S. Necibi. 1999. Beneficial insect attrac tion to light traps with different wavelengths. Bi ol. Control. 16: 185-188. Nutting, W. L. 1969. Flight and colony foundation. Pp 233-282. In K. Krishna and F. M. Weesner [eds.]. Biology of termites.Volume I. Academic Press, London and New York. 598p. Nutting, W. L. 1970. Composition and size of some termite colonies in Arizona and Mexico. Ann. Entomol. Soc. Am. 63: 1105-1110. Raina, A. Y. I. Park, and C. Florane. 2003. Behavior and reproductive biology of the primary reproductives of the Formosan Subterranean Termite (Isoptera: Rhinotermitidae). Sociobiology. 41: 37-48. Rebello, A. M. C., and C. Martius. 1994. Dispersal flights of termites in Amazonian forests (Isoptera). Sociobiology 24: 127-146. Rust, M. K., and R. J. Saran. 2006. Toxicity, repellency, and tran sfer of chlorfenapyr against subterranean termites (Isoptera: Rhinotermitidae). J. Econ. Entomol. 99: 864-872. Sakanoshita, A., and Y. ga. 1971. The influence of light on the emergencial mechanism of Formosan Termite, Coptotermes formosanus Shiraki. Jap. J. Appl. Entomol. Zool. 18: 143-150. Saran, R. J., and M. K. Rust. 2007 Toxicity, uptake, and transfer efficiency of fipronil in western subterranean termite (Isoptera: Rhinotermitidae). J. Econ. Entomol. 100: 495508. 52

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53 SAS. 2003. Version 9.1 SAS Institute, Cary, NC. Scheffrahn, R. H., P. Busey, J. K. Edwards, J. K e ek, B. Maharajh, and N-Y. Su. 2001. Chemical prevention of colony foundation by Cryptotermes brevis (Isoptera: Kalotermitidae) in attic modules. J. Econ. Entom. 94: 915-919. Scheffrahn, R. H., J. K e ek, R. Ripa, and P. Luppichini. 2008. Endemic origin and vast anthropogenic dispersal of the West Indi an drywood termite. Biol. Invasions doi: 10.1007/s10530-008-9293-3. Scheffrahn, R. H., N-Y. Su, J. K e ek, A. V. Liempt, B. Maharajh, and G. S. Wheeler. 1998. Prevention of colony foundation by Cryptotermes brevis and remedial control of drywood termites (Isoptera: Kalotermitidae) with selected chemical treatments. J. Econ. Entom. 91: 1387-1396. Shelton, T. G., J. E. Mulrooney, and T. L.Wagner. 2006. Tranfer of chlorfenapyr among workers of Reticulitermes flavipes (Isoptera: Rhinotermitidae) in the laboratory. J. Econ. Entomol. 99: 886-892. Snyder, T. E. 1926. The biology of the termite cast es. Quart. Rev. Biol. 1: 522-552. Su, N.-Y., and R. H. Scheffrahn. 1990. Economically important termites in the United States and their control Sociobiology 17: 77-94. Treacy, M., T. Miller, B. Black, I. Gard D. Hunt, and R. M. Hollingworth. 1994. Uncoupling activity and pesticide properties of pyrroles. Colloquium on the design of mitochondrial electron transport inhibitors as agrochemicals. Biochem. Soc. Trans. 22: 244. Wilkinson, W. 1962. Dispersal of alates and esta blishment of new colonies in Cryptotermes havilandi (Sjstedt) (Isoptera, Kalotermitidae). Bul. Entomol. Res. 53: 265-288. Williams, R. M. C. 1976. Factors limiting the distributi on of building-damaging dry-wood termites (Isoptera, Cryptotermes spp) in Africa. Mater. Organismen 3: 393-406. Zhao, X., V. L. Salgado, J. Z. YEH, and T. Narahashi. 2003. Differential actions of fipronil and dieldrin insecticides on GABA-gated chlo ride channels in cockroach neurons. J. Pharmacol. Exp. Therap. 306: 914-924.

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BIOGRAPHICAL SKETCH Maria Teresa Monteiro da Rocha Bravo Fe rreira was born in 1982, in Lisbon, Portugal. Born and raised in a big city, her love for insects did not arise until later in life. Between the years of 1994 and 2000, she attende d Liceu Cames, a very respected and traditional high school in Lisbon. There she devel oped her love for science by taking several different laboratory courses in chemistry a nd biology. In 2000, she graduated from High School and went on to be a freshman in college pur suing a biology major. At the Animal Biology Department at the Faculdade de Cincias da Un iversidade de Lisboa (Science College of the University of Lisbon) she joined the small entomology group in 2003 doing volunteer work in insect capturing and sorting. She did a year st udy in the Azorean Isle of So Miguel working with Diptera diversity in her fi nal year in college. She graduate d from college in July 2005, but continued her volunteer work with the entomology group until early 2006. In 2006, she moved to the Azorean Isle of Ter ceira where she began work as a technician on a project to determine management tools for the Cryptotermes brevis infestation in the Azores. She worked in this project until the end of that year. In 2007, she entered the graduate program at the University of Florida in the Department of Entomology and Nematology. She is continuing her studies and research at the Fort Lauderdale Research and Education Center. 59